Forced degradation of gliquidone and development of validated stability-indicating HPLC and TLC methods

Forced degradation studies of gliquidone were conducted under different stress conditions. Three degradates were observed upon using HPLC and TLC and elucidated by LC-MS and IR. HPLC method was performed on C18 column using methanol-water (85:15 v/v) pH 3.5 as a mobile phase with isocratic mode at 1 mL.min-1 and detection at 225 nm. HPLC analysis was applied in range of 0.5-20 µg.mL-1 (r =1) with limit of detection (LOD) 0.177 µg.mL-1. TLC method was based on the separation of gliquidone from degradation products on silica gel TLC F254 plates using chloroform-cyclohexane-glacial acetic acid (6:3:1v/v) as a developing system with relative retardation 1.15±0.01. Densitometric measurements were achieved in range of 2 -20 µg /band at 254 nm (r = 0.9999) with LOD of 0.26 µg /band. Least squares regression analysis was applied to provide mathematical estimates of the degree of linearity. The analysis revealed a linear calibration for HPLC where a binomial relationship for TLC. Stability testing and methods validation have been evaluated according to International Conference on Harmonization guidelines. Moreover, the proposed methods were applied for the analysis of tablets and the results obtained were statistically compared with those of pharmacopeial method revealing no significant difference about accuracy and precision.

本研究针对格列喹酮（gliquidone）开展了不同应激条件下的强制降解试验。采用高效液相色谱法（High Performance Liquid Chromatography，HPLC）与薄层色谱法（Thin Layer Chromatography，TLC）共检出三种降解产物，并通过液相色谱-质谱联用法（Liquid Chromatography-Mass Spectrometry，LC-MS）与红外光谱法（Infrared Spectroscopy，IR）完成结构解析。HPLC分析采用C18色谱柱，以甲醇-水（体积比85:15，pH 3.5）作为等度洗脱流动相，流速为1 mL·min⁻¹，检测波长为225 nm；该方法的线性范围为0.5~20 μg·mL⁻¹，相关系数r=1，检出限（Limit of Detection，LOD）为0.177 μg·mL⁻¹。TLC分析以硅胶TLC F254板为固定相，采用三氯甲烷-环己烷-冰乙酸（体积比6:3:1）作为展开系统，格列喹酮的相对比移值为1.15±0.01；薄层扫描测定的线性范围为每谱带2~20 μg，检测波长为254 nm，相关系数r=0.9999，检出限（LOD）为0.26 μg/谱带。本研究采用最小二乘回归分析对线性程度进行了数学表征，结果显示HPLC方法呈良好的线性校正曲线，而TLC方法则符合二项式关系。稳定性考察与方法验证均依据人用药品注册技术要求国际协调会议（International Conference on Harmonization，ICH）指南开展。此外，将所建立的两种分析方法应用于片剂样品的含量测定，并与药典方法的测定结果进行统计学比较，结果显示二者在准确度与精密度方面均无显著性差异。